Exhausted CD8⁺ T cell fate is programmed by dynamic CTCF-mediated enhancer activation and invariant CTCF-imposed...

Exhausted CD8⁺ T (T_EX) cells undergo extensive genome reorganization during differentiation, yet the drivers of this process remain elusive. Here we show that CTCF programmed CD8⁺ T_EX cell fates through two distinct modes of action. CTCF acquired de novo binding sites and concordantly induced open chromatin in early CD8⁺ T_EX cells responding to chronic viral infection. The dynamic CTCF binding activated enhancers and promoted chromatin looping. Consequently, genetic ablation of CTCF diminished chromatin accessibility and interaction strength, impairing CD8⁺ T_EX cell proliferation, effector function and bioenergetic mobilization. Conversely, invariant CTCF binding acted as essential chromatin barriers, and loss of CTCF disrupted insulation and caused aberrant chromatin self-association and undue RNA polymerase II pausing, leading to excessive activation of exhaustion- and stemness-linked genes. Thus, CTCF balanced CD8⁺ T_EX cell differentiation by gaining dynamic binding to induce cytotoxicity and sustain metabolic fitness, while its invariant binding compartmentalized exhaustion and stemness program genes to prevent their overexuberant activation.